Project Summary N-methyl-D-aspartate (NMDA) receptors have been associated with the development and progression of several neuropathologies including epilepsy and intellectual disabilities. When functioning optimally, NMDA receptors mediate a slow-component of fast excitatory neurotransmission to regulate processes of learning and memory. In response to agonist binding, the two glycine-binding GluN1 subunits and the two glutamate- binding GluN2 subunits (GluN2A-D) that typically comprise the NMDA receptor tetrameric assembly undergo conformational changes to induce channel pore opening and allow calcium and sodium to traverse the cell membrane. The stoichiometry with which these subunits are expressed within a given receptor impart distinct microscopic and macroscopic properties to that receptor, suggesting that each subunit within a tetramer makes a unique and independent contribution to receptor function. Moreover, each subunit is comprised of four semi- autonomous domains: the amino-terminal domain (ATD), the ligand binding domain (LBD), the transmembrane domain (TMD), and the carboxy-terminal domain (CTD). In a process referred to as gating, neurotransmitter binding induces cleft closure of the LBD and re-orientation of the M3 transmembrane helix that produces channel-pore opening. It has been suggested through structural analysis that the linker region between the LBD and the TMD contains features uniquely positioned to promote channel opening in response to ligand binding. A small two-turn helix within this linker, referred to as the pre-M1 helix, lies parallel to the lipid bilayer and is in van der Waals contact with a highly conserved region of the pore-forming M3 helix within the TMD. Additionally, several disease-associated de novo mutations within this region of the receptor have been identified and present dramatic effects on receptor function. In this study, whole-cell and single-channel patch- clamp electrophysiology and structure-based modeling will be used to elucidate the mechanism by which NMDAR function is dictated by subunit composition and the pre-M1 helix. The specific aims for the proposed study are 1) to determine the effects of a single copy of disease-associated mutations on single-channel properties, 2) to identify whether a conceptual model of subunit-specific conformational transitions can explain properties of channels with a single human mutation, and 3) to elucidate how pre-M1 interacts with the M3 and M4 helices to control NMDA receptor activation. The information gathered from the proposed experiments will provide insight into the effects and properties of disease-associated mutations, therapeutically relevant allosteric modulators, and receptors comprised of non-identical GluN2 subunits.